The critical step in the maintenance of normal hemostasis is the transformation of platelets from a non-adhesive to an adhesive state. Four proteins implicated in mediating platelet-platelet and platelet-vessel wall interactions are: fibrinogen (Fbg), fibronectin (Fn), thrombospondin (TSP), and von Willebrand factor (VWF). In addition, two putative platelet membrane receptors for these proteins have been identified: glycoprotein (GP) Ib and GPIIb/IIIa. The topographic relationship and vertical organization of these adhesive proteins and membrane receptors will be studied in three model systems: (i) isolated cytoskeletons of aggregated platelets, (ii) platelets stimulated in suspension, and (iii) platelets allowed to adhere to a variety of solid support. Initially, the distribution of retained membrane and adhesive glycoproteins in isolated cytoskeletons and intact, aggregated platelets will be evaluated by immunofluorescence of frozen thick sections, and by immunoelectronmicroscopy of frozen thin sections. We will test the hypothesis that receptors and adhesive proteins which mediate platelet aggregation cluster or patch at sites of platelet-platelet interaction in response to an aggregatory stimulus. These approaches will also be utilized to test the hypothesis that platelet activation by a stimulatory surface results in the polarized expression or redistribution of membrane, adhesive, and cytoskeletal proteins at sites of platelet-substratum interaction. In addition, we hypothesize that the nature of the surface influences that pattern of expression of these transmembrane complexes. The vertical organization of platelet attachments to various substrata including: collagen, Fn, laminin, and exposed endothelial cell matrix will be visualized by examining immunolabeled, perpendicular sections of adherent platelets. Particular attention will be paid to the distribution, orientation and interaction of platelet adhesive proteins (Fbg, Fn, TSP and VWF) with platelet membrane proteins (GPIb and GPIIb/IIIa) and platelet cytoskeletal proteins (actin, myosin, actin binding protein, alpha-actinin and vinculin) at sites of platelet-substratum interaction.